Pluripotent stem cells are cells having both a self-renewal ability to renew themselves and pluripotency. In recent years, a transplantation therapy using cells differentiated from pluripotent stem cells has attracted attention in regenerative medicine aimed at regeneration and functional recovery of cells, tissues, and organs with malfunction or dysfunction. In addition, the cells differentiated from pluripotent stem cells are expected to be applied to, for example, drug toxicity tests. As described above, it is a big issue in a pharmaceutical field to induce differentiation of pluripotent stem cells into cells of interest and amplify the cells of interest.
Induced pluripotent stem cells (hereinafter sometimes abbreviated as iPS cells) and embryonic stem cells (hereinafter sometimes abbreviated as ES cells) are known as pluripotent stem cells. Those cells are each expected to be applied to, for example, regenerative medicine based on a transplantation therapy or the like involving inducing differentiation into cells of interest and using the cells of interest, and drug toxicity tests.
For example, when hepatocytes can be induced to differentiate from human iPS cells or human ES cells, it is expected to develop a transplantation therapy to the cases of liver failure. Liver is constructed mainly of hepatocytes which are hepatic parenchymal cells, hepatic nonparenchymal cells such as bile duct epithelial cells, and the like. The liver is an organ which secretes bile, filters and detoxifies absorbed nutrients, metabolizes drugs, stores sugars, and regulates blood glucose, and besides, produces fibrinogen, heparin, an anti-anemic substance, and the like, and thus is an organ essential for life. Therefore, liver failure, in which the number of functioning hepatocytes excessively decreases, is a fatal pathological condition. In the cases of liver failure, there occurs an extremely severe condition such as bleeding tendency due to an excessive lack of coagulation factors, or hepatic coma. Transplantation of the hepatocytes can serve as a radical therapy for such pathological condition.
The inventor of the present invention provided a method to induce differentiation of ES cells into hepatocytes and obtain a cell culture product substantially composed of hepatocytes from a cell group containing the differentiated hepatocytes (Japanese Patent No. 4759723 and Japanese Patent Application Laid-open No. 2005-253374). Specifically, the inventor focused attention on the fact that the hepatocytes had a series of enzymes involved in glycolysis, a urea cycle, and the like, and hence survived in a medium containing galactose and ornithine that were added as substrates, but not containing glucose and arginine that were produced by the series of enzymes, and provided a method of producing a cell culture product substantially composed of hepatocytes through culture of cells differentiated from ES cells in such medium in which cells differentiated into other cells than hepatocytes died.
However, application of human ES cells to humans involves immune rejection due to the fact that the ES cells are embryonic stem cells derived from others, and an ethical problem.
Meanwhile, iPS cells, which are pluripotent stem cells induced from somatic cells such as human fibroblasts by a gene recombinant technology, are capable of being produced from autologous cells. Thus, the iPS cells hardly cause a problem such as immune rejection, and are expected to be applied to regenerative medicine (Takahashi, K. et al., “Induction of Pluripotent Stem Cells from Adult Human Fibroblasts by Defined Factors,” Cell, 2007, Vol. 131, No. 5, p. 861-872).
As a technology for inducing differentiation of human iPS cells into hepatocytes, there is a report on, for example, a method in which growth factors and/or transcription factors for promoting differentiation to hepatocytes were each added and introduced to culture of human iPS cells (Tomizawa, M. et al., “Liver”, 2011, Vol. 52, (Suppl. 2): A680, Inamura et al., “Efficient Generation of Hepatoblasts from Human ES Cells and iPS Cells by Transient Overexpression of Homeobox Gene HEX,” Molecular Therapy, 2011, Vol. 19, No. 2, p. 400-407, and Tomizawa, M. et al., “Single-step protocol for the differentiation of human-induced pluripotent stem cells into hepatic progenitor-like cells,” Biomedical Reports, 2013, Vol. 1, p. 18-22 (published online on Monday, Aug. 13, 2012 as Doi: 10.3892/br.2012.2)).
There is also a report on a method of forming liver from human iPS cells (Takebé, T. et al., “Vascularized and functional human liver from a iPSC-derived organ bud transplantation,” Nature, 2013, Vol. 499, p. 481-489). This method is a method of forming liver, comprising firstly promoting differentiation from iPS cells into hepatocytes in vitro, mixing the resultant with vascular endothelial cells and mesenchymal stem cells, and then transplanting the mixture into a mouse brain. Such a method is required to have no exposure to foreign proteins when applied to humans. In addition, the iPS cells that were promoted to differentiate into hepatocytes die when they are not transplanted into the mouse brain, but are maintained in vitro. Further, it takes several weeks to induce differentiation to hepatocytes in vitro by such method, and besides, mature hepatocytes cannot be obtained (Si-Tayeb, K. et al., “Highly efficient generation of human hepatocyte-like cells from induced pluripotent stem cells,” Hepatology, 2010, Vol. 51, p. 297-305). Accordingly, in order to achieve the application to humans, it is essential to develop a method of producing hepatocytes from iPS cells in a short period of time without any exposure to foreign proteins.
As described above, the iPS cells are expected to be applied to regenerative medicine. However, when inducing differentiation of iPS cells into cells of interest in vitro, the resultant obtained is a cell group containing undifferentiated iPS cells as well as the cells of interest, and hence there is a risk in that the remaining undifferentiated iPS cells form a tumor through transplantation (Cunningham et al., “Lessons from human teratomas to guide development of safe stem cell therapies,” Nature Biotechnology, 2012, Vol. 30, p. 849-8.57). Accordingly, it is necessary to separate differentiation-induced cells from a cell group containing the differentiation-induced cells and undifferentiated iPS cells, in order to utilize cells induced to differentiate from iPS cells in regenerative medicine, such as transplantation.
The inventor of the present invention developed and reported a method of collecting only human primary cultured hepatocytes from co-culture of human iPS cells with human primary cultured hepatocytes by killing the human iPS cells, and hepatocyte selection medium (HSM) to be used in the method (Tomizawa, M. et al., “Survival of primary human hepatocytes and death of induced pluripotent stem cells in media lacking glucose and arginine,” PLoS One, 2013, Vol. 8, e71897 and Japanese Patent Application No. 2012-286978). This medium is prepared by adding galactose and ornithine, but not adding glucose and arginine that are essential for cell survival. The hepatocytes have a series of enzymes involved in gluconeogenesis and a urea cycle, and hence can synthesize glucose and arginine. Thus, the hepatocytes can survive even when cultured in HSM. However, the human iPS cells lack gluconeogenesis and the urea cycle, and die in 3 days when cultured in HSM. Therefore, when a method of producing hepatocytes from human iPS cells is established, the remaining undifferentiated human iPS cells can be removed from a produced cell group by culturing the cell group in HSM for 3 days, and a cell culture consisting substantially of hepatocytes can be obtained. HSM is constructed only of ingredients contained in a general medium, and thereby has an extremely low risk of damaging the resultant hepatocytes.